HIV (Human Immunodeficiency Virus) is the virus responsible for the acquired immunodeficiency syndrome (AIDS), and belongs to the human retrovirus family. AIDS is now considered as one of the greatest threats to human health. An HIV-infected individual can transmit the disease, although remain asymptomatic for years.
The suspected etiological agent responsible for AIDS was independently identified in 1983-1984 by several research groups (see e.g. Barre-Sinoussi et al., Science 220:868-871; Montagnier et al., in Human T-Cell Leukemia Viruses (Gallo, Essex & Gross, eds.); Vilmer et al., The Lancet 1:753), and HIV nomenclature was subsequently unified.
The HIV family comprises several types and subtypes. HIV1 viruses can be classified according to subtypes. Examples of HIV1 sub-types include HIV1-M and HIV1-O. Similarly, HIV2 viruses encompass various sub-types, e.g. HIV2-A and HIV2-B.
For drug development assays, prophylaxis, as well as for treatment of AIDS, it has now become of great importance to be able to quickly and easily identify and quantify the group(s), type(s) and subtype(s) of HIV viruses present in a given sample.
By HIV group, we herein understand any HIV group, irrespective of it being known at the priority date or not. Various HIV groups are known in the art, and are described in the corresponding literature and databases, e.g. ncbi on the internet. Examples thereof include HIV1-M and HIV1-O.
By HIV subtype, we herein understand any HIV subtype, irrespective of it being known at the priority date or not. Various HIV subtypes are known in the art, and are described in the corresponding literature and databases, e.g. ncbi on the internet.
By HIV isolate, we herein understand any HIV isolate or strain, irrespective of it being known at the priority date or not. Various HIV isolates are known in the art, and are described in the corresponding literature and databases, e.g. ncbi on the internet. Some isolates are regarded as references. Examples thereof include K03455, L20587 and M30502.
A possible approach could rely on the development of specific antibodies. However, in terms of sensitivity and specificity, a PCR-based approach usually looks very promising. Also, it generally offers the possibility to work on small samples.
Amplification methods, especially polymerase chain reaction (PCR) and PCR-based methods, e.g. reverse-transcriptase PCR (RT-PCR) and PCR are known in the art (Molecular Cloning: A Laboratory Manual, Maniatis, Fritsch, and Sambrook, CSHL Press; Molecular Biology of the Cell, Alberts et al.; PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, CSHL Press; The Polymerase Chain Reaction, Mullis, Ferré, and Gibbs, Birkhauser Boston Press; Gene quantification, Ferré, Birkhauser Boston Press.)
These methods are generally very efficient tools for the qualitative and quantitative analysis of complex biological samples.
However, the efficiency of these techniques typically crucially depends on the design and the choice of primers.
There is prior art describing HIV1-specific primers (U.S. Pat. No. 5,712,385; EP 1 043 407; WO 03/020878; EP 1 344 837). There is also prior art describing HIV2-specific primers (U.S. Pat. No. 5,962,665).
Depending upon the working conditions, at least some of these prior art primers may show a sufficient HIV1 or HIV2 specificity, thereby allowing for a specific detection of HIV1 and HIV2. However, to the applicant's knowledge, none of them allows for:                a real-time quantitative specific detection of said subtypes, or for        a detection of said subtypes in multiplex which would remain specific, or for        a detection of said subtypes in multiplex which would remain quantitative, even when implemented in real-time.        
Such a real-time quantitative multiplex specific detection would however be more reliable and informative on the patient's actual infection stage. It would thus give access to a more accurate diagnosis, and as a consequence, would allow to more accurately balance the positive against the deleterious effects of a given treatment. It would allow adjusting or choosing the treatment which should be the most efficient to the particular patient being diagnosed.
Such a real-time quantitative multiplex specific detection would also have the advantage of being faster and easier to run, especially on a large scale.